A nucleocapsid (hereinafter, referred to as ‘NC’ protein of HIV (human immunodeficiency virus) plays a structural role in virus assembly, as well as a functional role in viral life cycles, which are described as follows. First, the NC protein is involved in viral genomic encapsidation, which is attributed to two zinc finger domains consisting of a distinctive Cys-X2-Cys-X4-His-X4-Cys motif (CCHC motif). It has been known that the domain shows a high conservation in all retroviruses, and is essential for HIV RNA packaging and infectious virus production. Second, the NC protein has been known to promote the annealing of tRNA primer and strand transfer during viral reverse transcription (RT). From this, it can be seen that the NC protein plays a crucial role in viral replication. Third, the NC protein has nucleic acid chaperone activity required for viral life cycle. Further, the NC protein has been recently reported to play a specific role in the insertion of viral DNA into host chromosomes. Accordingly, it can be said that the studies on the NC protein is very important with respect to clarifying biological functions of the NC protein in HIV life cycle and the development of effective antiviral agents against crucial HIV proteins.
A prerequisite for understanding the biological functions of the NC protein in vivo is the development of effective methods for expressing the NC protein in animal cells. However, the expression of other structural proteins of HIV may be restricted in animal cells, since viral codon usage is different from that of animal cells or some of the viral genes contains what is known as an inhibitory sequence (INS). There was a problem in that the NC protein is hardly expressed in animal cells, even though the NC protein contains no INS, unlike other structural proteins of HIV. Accordingly, most studies on the NC protein have been performed by using a recombinant NC protein expressed in E. coli or by using genetic analysis.
Meanwhile, there are several examples that the level of heterologous expression is enhanced by the replacement of rare codons with those preferred by the host (codon optimization). For example, it has been reported that BPV (Bovine papillomavirus) late genes L1 and L2 are codon-optimized for the mammalian codon usage pattern, leading to increase in their expression levels in mammalian cell (Cos-1) culture, as compared to those sequence of wild type HPV (Zhou, et al., J. Virol. 73, 4972-4982, 1999). In this work, every BPV codon which occurred more than twice as frequently in BPV than in mammals (ratio of usage>2), and most codons with a usage ratio of >1.5 were conservatively replaced by the preferentially used mammalian codon. In the PCT application of WO 97/31115, WO 97/48370 and WO 98/34640 (Merck & Co., Inc.), codon optimization of HIV genes or segments thereof has been shown to result in increased protein expression and improved immunogenicity when the codon-optimized sequences are used as DNA vaccines in the host mammal for which the optimization was tailored.
The present inventors have kept the above-mentioned points in mind, and made an effort to develop a method for producing an NC protein of HIV capable of overcoming the above-described problems. We have found, however, that a wild type NC protein can not be expressed by codon-optimization alone, and that a wild type NC protein can be expressed by additionally linking an intron sequence and an mRNA stability element in the upstream and downstream of NC gene respectively and, with such RNA optimization, its expression efficiency is greatly improved, thereby completing the present invention.